The Effect Of MiR-18a On Autophagy Via MTOR Pathway In C6 Glioma Cells

Objective: To investigate the effect of miR-18 a on autophagy in C6 glioma cells and its potential molecular mechanism.Methods:1.The transient transfection method was used to construct cell models of miR-18 a overexpression and miR-18 a downregulation,and the transfection efficiency was detected by observing the fluorescence intensity of fluorescence-labeled(FAM)miRNA negative control transfected cells under the inverted fluorescence microscope.In addition,the expression of miR-18 a was analysed by using RT-PCR after transfecting C6 cells with miR-18 a mimics or miR-18 a inhibitor.2.The effect of miR-18 a on C6 glioma cell proliferation was measured by CCK-8 assay.3.The EGFP-m Cherry-LC3 plasmid was used to instantaneously transfect cells in each group,and the fluorescence intensity of EGFP/m Cherry-LC3 was observed under inverted fluorescence microscope to dynamically monitor the autophagy flux in each group.4.Western blot was used to detect the effect of miR-18 a on the expression of autophagyrelated proteins LC3,p62 and Beclin-1 in C6 cells,as well as to measure the protein expression of LC3 and p62 in C6 cells treated with chloroquine and rapamycin.The up-regulation and down-regulation of miR-18 a on the expression of key proteins of mTOR signaling pathway in C6 glioma cells were further examined.The effects of down-regulation of miR-18 a on the expression of NF-κB and mTOR pathway proteins in TNF-α-treated C6 cells were also detected.5.RT-PCR was used to detect the effects of up-regulation and down-regulation of miR-18 a on the m RNA expression levels of Beclin-1,LC3,and mTOR in C6 cells.The expression levels of miR-18 a and mTOR m RNA in C6 glioma cells treated with TNF-α were also examined by RT-PCR.Results:1.C6 cells were transfected with negative control of FAM-labeled miRNA,and the number of cells successfully transfected under fluorescence field/number of cells with bright field in the same field was >80% under inverted fluorescence microscope.RT-PCR results showed that the expression of miR-18 a in C6 glioma cells in the miR-18 a mimics group was markedly increased compared with the blank group and NC mimics group(P<0.05).Compared with the blank group and NC inhibitor,the expression of miR-18 a in C6 glioma cells in the miR-18 a inhibitor group was significantly decreased(P<0.05).2.Knockdown of miR-18 a significantly inhibited the proliferation of C6 glioma cells.3.Autophagic flux assay showed that inhibition of miR-18 a inhibited autophagosome and lysosome fusion and blocked autophagic flux as observed under inverted fluorescence microscopy.The expression levels of LC3-II/LC3-Ⅰ were significantly higher in the overexpression of miR-18 a C6 cells than in the control group after treatment with the autophagy late inhibitor chloroquine,indicating that miR-18 a overexpression promoted the formation of autophagosomes and enhanced autophagic flux.4.The effects of miR-18 a on the expression of autophagy-related genes and proteins expression in C6 glioma cells: Knockdown of miR-18 a inhibited the m RNA expressions of Beclin-1 and LC3 in C6 glioma cells(P<0.05).Overexpression of miR-18 a promoted the m RNA expressions of Beclin-1 and LC3 in C6 glioma cells(P<0.05).Down-regulation of miR-18 a significantly decreased the protein expression levels of LC3-II/LC3-Ⅰ and Beclin-1 and significantly increased the expression of p62 protein in C6 glioma cells(P<0.05).Up-regulation of miR-18 a significantly increased the expression levels of LC3-II/LC3-Ⅰ protein and significantly decreased the expression of p62 protein in C6 glioma cells(P<0.05).5.The effect of miR-18 a on mTOR signaling pathway in C6 glioma cells: The expression level of mTOR m RNA was significantly increased in C6 glioma cells by downregulation of miR-18 a.In addition,knockdown of miR-18 a promoted the expression of p-mTOR,p-p70S6 K and p-4EBP1 proteins in C6 glioma cells,but had no significant effect on the expression levels of mTOR,p70S6 K and 4EBP1 total proteins.This demonstrated that downregulation of miR-18 a could activate the mTOR signaling pathway.The expression level of mTOR m RNA was significantly reduced in the overexpression of miR-18 a of C6 cells.Meanwhile,upregulation of miR-18 a significantly inhibited the expression of p-mTOR,p-p70S6 K and p-4EBP1 proteins in C6 glioma cells,while it had no significant effect on the expression levels of mTOR,p70S6 K and 4EBP1 total proteins.This indicated that overexpression of miR-18 a could inhibit the mTOR signaling pathway.6.The protein expression level of LC3-II/LC3-Ⅰ was significantly decreased compared with the control group after treatment of miR-18 a down-regulated C6 cells with rapamycin(P<0.05).This indicated that downregulation of miR-18 a could reduce rapamycin-induced autophagy in C6 glioma cells7.TNF-α promoted the expression of miR-18 a and activated NF-κB and mTOR signaling pathways in C6 glioma cells.In addition,inhibition of miR-18 a could both inhibit NF-κB signaling pathway and enhance the TNF-α-mediated activation of NF-κB and mTOR pathways.Conclusion: miR-18 a could regulate autophagy in glioma cells via regulating the mTOR pathway.In addition,there might be a positive feedback loop between the NF-κB pathway and miR-18 a,and this positive feedback loop may play an important role in the malignant progression of glioma.Therefore,miR-18 a may be a potential therapeutic target in glioma.